The suprachiasmatic nucleus (SCN) in the mammalian brain contains the master circadian clock. Individual SCN neurons are competent circadian oscillators and constitute a neural network that stabilizes and enhances the generation of circadian timing signals. 3-Aminobutyric acid (GABA) shifts the phase of the circadian clock, inhibits light-induced phase shifts, synchronizes the activity of dorsal and ventral SCN neurons, and modulates the action potential firing frequency of individual SCN neurons. The long-term goal of our research is to understand the role of GABA-mediated neurotransmission in the generation of circadian timing signals. The strength and functional consequences of GABAergic neurotransmission are regulated by the intracellular Cl- concentration. In the adult brain, activation of GABAA receptors inhibits a majority of SCN neurons during the day, while at night the majority of GABAA responses are excitatory. The day-night difference in GABAA receptor-mediated synaptic activity results from dynamic regulation of the intracellular Cl- concentration during the circadian cycle. The sodium-potassium-chloride (NKCC1) cotransporter responsible for increasing intracellular Cl- and the potassium-chloride (KCC) family of cotransporters that reduce intracellular Cl- are heterogeneously expressed throughout the SCN. We hypothesize that the circadian variation in GABA neurotransmission depends on the type and activity of the cotransporters expressed in individual SCN neurons. The goal of this application is to identify the mechanisms by which the circadian clock regulates the strength and polarity of GABA-mediated signaling in the SCN and the role this plays in regulating the SCN neural circuit. We will use a combination of electrophysiological and imaging techniques to determine the role of NKCC1 and the KCC family of cotransporters in mediating the magnitude and diversity of GABAergic transmission in the SCN. The Specific Aims of the proposal are: 1) Determine, in individual SCN neurons, whether the direction of the GABA-activated chloride current underlies the shift in GABA response from inhibitory to excitatory during a circadian cycle. 2) Investigate the role of the NKCC1 and KCC cotransporters in regulating the activity of the SCN neural network. 3) Examine the contribution of the NKCC1 cotransporter in determining the balance between inhibitory and excitatory GABAA receptor- mediated currents in phenotypically identified SCN neurons. 4) Examine the role of Cl- efflux as regulated by KCC cotransporters in modulating the magnitude and polarity of GABAA receptor-mediated synaptic transmission. PUBLIC HEALTH RELEVANCE: Humans live in a complex 24-hour-a-day society where desynchrony between circadian clocks and the environment contributes to an increased vulnerability to a variety of diseases. The current application aims to identify the neuronal mechanisms that lead to the generation of circadian timing signals. This knowledge will guide the development of new strategies for the mitigation or correction of circadian based disorders.